1. Field of the Invention
The invention relates to a test field for a test carrier for the determination of an analyte from whole blood with the aid of a reagent system contained in the test carrier, said reagent system comprising a colour formation reagent, with a test field which has a sample application side, to which the blood sample is fed, and a detection side on which, as a result of reaction of the analyte with the reagent system, an optically detectable change occurs, the test field being designed so that erythrocytes contained in the sample do not gain access to the detection side.
2. Description of the Related Art
For the qualitative or quantitative analytical determination of constituents of blood, so-called carrier-bound tests are increasingly being used. In these, a reagent system is embedded in at least one test field of a solid test carrier, said test field consisting of a single layer or a plurality of layers, said test carrier being brought into contact with the sample. The reaction of sample and reagent system gives rise to an optically detectable change, in particular to a colour change which can be evaluated visually or with the aid of an instrument, usually by reflection photometry. Instead of leading to a colour change, the reaction may also give rise to the occurrence of, or to a change in, some other optically detectable signal, for example, fluorescence or luminescence.
Test carriers (which in the English-language literature are frequently also called "solid reagent analysis elements") are often designed as test strips, which essentially consist of a longitudinal carrier layer made of a plastic material, and a single test field or a plurality of test fields applied to it. However, test carriers are also known in which, analogously to a photographic slide, a test field is surrounded by a plastic frame.
Carrier-bound tests are characterised in particular in that they are easy to use. It is all the more unfortunate that, in the majority of test carriers known to date, the blood cannot be used directly as whole blood. Rather, it is necessary to separate the red blood corpuscles (erythrocytes) in order to obtain colourless plasma or serum. This is usually done by centrifuging. However, this involves an additional operation requiring a relatively large amount of blood and expensive equipment.
Many attempts have therefore been made to provide test carriers which permit analytical determinations to be made directly from blood. One can distinguish two fundamentally different possible solutions.
In the first attempted solution, evaluation of the colour change visually or with the use of equipment takes place on the same side of the test field as that to which the sample is applied. In this arrangement, the test field is constructed so that the analyte from the sample penetrates to the reagents through the test field surface, whereas the erythrocytes are retained. After a defined time, the blood sample is wiped or washed from the test field surface and the colour change is observed. Examples of such test carriers are described in U.S. Pat. No. 3,630,957 and in EP-B-130 335 and EP-A-0 217 246.
In the second attempted solution, the sample is applied to one side of the test field (sample application side), and the colour change on the other side (detection side) is recorded.
A major advantage of this method of detection is that the blood need not be wiped or washed off. For this reason, these test carriers are also called non-wipe ("NW")-test carriers.
With the wiping operation there is eliminated not only a tedious operation, but also a possible source of error which may result from imprecise observance of the point in time at which the blood must be removed. However, this attempted solution is especially difficult to embody. An erythrocyte or blood pigment filter is required which on the one hand reliably retains the intensely colouring constituents of blood but on the other hand allows the analyte to pass through completely and sufficiently rapidly. It is proving extraordinarily difficult to find a test field composition which meets these requirements.
In U.S. Pat. No. 3,663,374 and U.S. Pat. No. 4,256,693, a membrane filter is used. Although membrane filters are in principle suitable for filtering off erythrocytes, their use in test carriers has not proved successful. The same applies to the combination of the membrane filter with a glass-fibre layer also disclosed in these U.S. patents, said glass-fibre layer being intended to prevent blockage of the membrane filter with coarser particles. The manufacture of test carriers of this type would be very expensive, without satisfactory functioning being achieved.
U.S. Pat. No. 4,069,017 and a plurality of patents from the same applicant also address the possibility of providing, in a test carrier, an intermediate layer preventing the passage of erythrocytes, said intermediate layer at the same time containing radiation-blocking constituents to ensure that the light rays of the evaluation instrument cannot penetrate the erythrocyte-containing layer. However, this specification contains no details as to how the intermediate layer could be structured in the individual case in order to achieve the filtering of erythrocytes.
U.S. Pat. No. 4,824,639 describes the use of an asymmetrical membrane, such as that known for industrial separation procedures (reverse osmosis), for the purposes of erythrocyte separation. The membrane is manufactured in a coagulation procedure, by immersing a partially or completely gelatinised layer into a coagulation bath, and may also contain reagents. In this arrangement, the optical evaluation is said to be possible both from the blood application side (after wiping) and from the opposite side.
In EP-A-0 302 287 a test field is disclosed which has a composite layer which is manufactured by applying, in a liquid form, a detection layer containing the colour formation reagent on to a base layer which faces the sample application side. The base layer contains a polymeric film former, diatomaceous earth and a pigment. The detection layer contains a polymeric film former which, during application in a liquid form, partially penetrates the base layer and forms a transition zone in which the erythrocytes are retained.
The problem caused by the pigment constituents of the blood is further exacerbated by the fact that the capillary blood usually used, which is obtained by puncturing the skin, is inevitably partially haemolysed owing to the associated mechanical stresses, i.e., it contains blood pigment liberated from erythrocytes which have been destroyed. Although the haemolysed proportion is generally smaller than 0.1%, owing to its intense colour the liberated blood pigment still causes measuring inaccuracies even when the erythrocytes have been separated completely.
Since none of the previously known embodiments of an NW test carrier with erythrocyte separation exhibits satisfactory properties in every respect, in a commercially available embodiment of such a test carrier, separation of erythrocytes has been completely dispensed with, and distortion due to the intense red colour has been compensated for in the measuring technique with the aid of measurement at two different wavelengths. However, this impairs the measuring accuracy because the dark colour of the blood substantially reduces (by about a half) the signal difference, that is, the difference in the measured diffuse reflection within the measuring range of the test. Furthermore, the cost of instrumental evaluation is greatly increased, and visual checking of the colour change is impossible.